Rabbit IgG ELISpotBASIC kit (ALP) (CAT#: ITS-0322-P38)

4 plates

1.Vial 1(green top):Monoclonal antibody MT5131 (1.2 ml);Concentration:0.5 mg/ml.
2.Vial 2(yellow top):Biotinylated monoclonal antibody MT536 (50 ul);Concentration:0.5 mg/ml.
3.Vial 3 (white top):Streptavidin-Alkaline Phosphatase (50 pul).
4.Antibodies are supplied in sterile filtered (0.2 um)PBS with 0.02% sodium azide. Strepta-vidin-ALP is supplied in 0.1 M Tris buffer with 0.002% Kathon CG.Vials have beenoverfilled to ensure recovery of the specified amount.

200 µL

2h

Pre-coated

1.Dilute the coating mAb MT5131 to 15 ug/ml in sterile PBS, pH7.4.
2.PVDF plates need to be treated (activated) with ethanol before coating.
3.Wash plate 5 times with sterile water, 200 ul/well.
4.Add 100 ul/well of the antibody solution and incubate overnight at 4-8°°C.
5.Wash plate 5 times with sterile PBS, 200 ul/well, to remove excess antibody.
6.For blocking, add 200 ul/well of medium containing 10% of the same serum as used for the cell suspensions.Incubate for at least 30 minutes at room temperature.
7.Remove the medium and add the cell suspension to the ELISpot plate ( 100 ul/well).
8.Put the plate in a 37°Chumidified incubator with 5% CO, and incubate for 16-24 hours.
9.To remove the cells, empty the plate and wash 5 times with PBS, 200 ul/well.
10.Dilute biotinylated antigen to a suitable con-centration (0.01-1 ug/ ml) in PBS-0.5% fetalcalf serum (FCS).Add 100 ul/well and incu-bate for 2 hours at room temperature.
11.Wash plate as above (step C1).
12.Dilute the Streptavidin-ALP(1:1000) in PBS-0.5% FCS and add 100 ul/well. Incubate for 1 hour atroom temperature.
13.Wash plate as above(step C1).
14.Add 100 ul/well of substrate solution (e.g.BCIP/NBT) and develop until distinct spots emerge.
15.Stop colour development by washing extensively in tap water. If desirable, remove the plate from theplate tray or the underdrain and rinse the underside of the membrane.
16.Leave the plate to dry. Inspect and count spots in an ELISpot reader or in a dissection microscope.
17.Store plate in the dark at room temperature.

Rabbit

Capture mAb (MT5131),Biotinylated detection mAb (MT536),Streptavidin-ALP

We recommend the use of PVDF-based membrane plates. Maximal antibody binding capacity of these platesis obtained by a brief treatment with ethanol.

PBS for washing and dilution should be filtered (0.2 um) for optimal results.Although possible to use, we donot recommend the inclusion of Tween or other detergents in the washing and incubation buffers.

4°C-8°C

Plates should be kept at room temperature.

The expiry date indicates how long unopened products, stored according to instructions, are recommended for use.

The serum should be selected to support cell culture and give low background staining. We recom-mend the use of fetal calf serum. Alternatively serum-free medium evaluated for cell culture can beused.

For Research Use Only. Not for use in diagnostic procedures.

1.Antibody coating:Cytokine-specific monoclonal capture antibodies are immobilized on an ethanol-treated PVDF membrane plate.
2.Cell incubation:Cells are added to the wells in the presence or absence of activating stimuli, and then incubated to allow for cytokine secretion.
3.Cytokine capture:Secreted cytokines bind to the capture antibodies on the membrane immediately surrounding the activated cells.
4.Detection antibodies:Following removal of the cells and washing of the plate wells, biotinylated cytokine-specific detection antibodies are added to the wells.
5.Streptavidin-enzyme conjugate:To enable the formation of spots on the membrane, a streptavidin-enzyme conjugate is added to the wells.
6.Addition of substrate:Colorimetric substrate is added to the wells and will form an insoluble precipitate when catalyzed by the enzyme; a visible representation of cytokine release by a single activated cell.
7.Analysis Spots are counted in an automated ELISpot reader or under a dissection microscope, and the frequency of secreting cells is calculated.